1. Photoaffinity labeling with thyroid hormone (using (I125) and (I131) T4 revealed that the T4-binding proteins present in nuclear extract, plasma membrane and cytosol differ in their molecular masses and showed different inhibition of incorporated radioactivity in the presence of a 1000-fold molar excess of unlabeled hormone, thus indicating different hormone-binding activities among these T4 binding proteins. 2. Photoaffinity labeling with underivatized (I125)thyroxine using light above 300 nm, confirmed the presence in the high salt nuclear extract (0.4 M NaCl) of two proteins (56 and 45 kDa) which bind the hormone with high affinity. Since the 56/45 kDa ratio was considerably higher than in previous experiments in which much longer irradiation times were used (30 min. vs. 2 min.), it appears likely that the 45 kDa protein is a breakdown product derived from the 56 kDa protein.